Production of ultra-high-molecular-weight poly-γ-glutamic acid by a newly isolated Bacillus subtilis strain and genomic and transcriptomic analyses.

Poly-γ-glutamic acid (γ-PGA) is a microbial-derived polymer with molecular weight (Mw) from 104 to 107 Da, and the high-Mw (> 7.0 × 105 Da) or ultra-high-Mw (> 5.0 × 106 Da) γ-PGA has important application value as a tissue engineering material, as a flocculant, and as a heavy metal remover. Therefore, how to produce these high-Mw γ-PGAs with low cost and high efficiency has attracted wide attention. In this study, a γ-PGA producer was isolated from the natural environment, and identified and named Bacillus subtilis GXD-20. Then, the ultra-high-Mw (> 6.0 × 106 Da) γ-PGA produced by GXD-20 was characterized. Interestingly, GXD-20 could produce γ-PGA at 42°C, and exhibited a γ-PGA titer of up to 22.29 ± 0.59 g L-1 in a 5-L fermenter after optimization of the fermentation process. Comparative genomic analysis indicated that the specific protein sequence and subcellular localization of PgdS (a γ-PGA-degrading enzyme) were closely related to the ultra-high-Mw of γ-PGA. Transcriptomic analysis revealed that the high γ-PGA titer at 42°C was mainly related to the high expression of genes encoding enzymes for sucrose transportation and utilization, nitrogen transportation, endogenous glutamate synthesis, and γ-PGA synthesis. These results provide new insights into the production of ultra-high-Mw γ-PGA by Bacillus at high temperatures.

Enhancing Poly-γ-glutamic Acid Production in Bacillus tequilensis BL01 through a Multienzyme Assembly Strategy and Expression Features of Glutamate Synthesis from Corynebacterium glutamicum.

The enhancement of intracellular glutamate synthesis in glutamate-independent poly-γ-glutamic acid (γ-PGA)-producing strains is an essential strategy for improving γ-PGA production. Bacillus tequilensis BL01ΔpgdSΔggtΔsucAΔgudB:P43-ppc-pyk-gdhA for the efficient synthesis of γ-PGA was constructed through expression of glutamate synthesis features of Corynebacterium glutamicum, which increased the titer of γ-PGA by 2.18-fold (3.24 ± 0.22 g/L) compared to that of B. tequilensis BL01ΔpgdSΔggtΔsucAΔgudB (1.02 ± 0.11 g/L). To further improve the titer of γ-PGA and decrease the production of byproducts, three enzymes (Ppc, Pyk, and AceE) were assembled to a complex using SpyTag/Catcher pairs. The results showed that the γ-PGA titer of the assembled strain was 31.31% higher than that of the unassembled strain. To further reduce the production cost, 25.73 ± 0.69 g/L γ-PGA with a productivity of 0.48 g/L/h was obtained from cheap molasses. This work provides new metabolic engineering strategies to improve the production of γ-PGA in B. tequilensis BL01. Furthermore, the engineered strain has great potential for the industrial production of γ-PGA from molasses.

Electrofermentation increases concentration of poly γ-glutamic acid in Bacillus subtilis biofilms.

Fluctuations in redox conditions in bioprocesses can alter the end-products, reduce their concentration, and lengthen the process time. Electrofermentation enables rapid metabolic modulation of biosynthesis and allows control of redox imbalances in biofilm-based fermentation processes. In this study, electrofermentation is used to boost the production of the bacterial biopolymer poly-γ-glutamic acid (γ-PGA) from Bacillus subtilis ATCC 6051. When compared to control experiments (3.3 ± 0.99 g L-1 ), the application of an electrode potential E = 0.4 V versus Ag/AgCl results in a more than two-fold increase in the production of γ-PGA (9.13 ± 1.4 g L-1 ). Using an engineered B. subtilis strain, in which γ-PGA production is driven by isopropyl ß-d-1-thiogalactopyranoside, electrofermentation improves polymer concentrations from 15.4 ± 1.5 to 23.1 ± 1.6 versus g L-1 . These results confirm that electrofermentation conditions can be adopted to increase the concentration of γ-PGA and perhaps other extracellular biopolymers in industrial strains.

Effective Removal of Different Heavy Metals Ion (Cu, Pb, and Cd) from Aqueous Solutions by Various Molecular Weight and Salt Types of Poly-γ-Glutamic Acid.

In light of industrial developments, water pollution by heavy metals as hazardous chemicals has garnered attention. Addressing the urgent need for efficient heavy metal removal from aqueous environments, this study delves into using poly-γ-glutamic acid (γ-PGA) for the bioflocculation of heavy metals. Utilizing γ-PGA variants from Bacillus subtilis with different molecular weights and salt forms (Na-bonded and Ca-bonded), the research evaluates their adsorption capacities for copper (Cu), lead (Pb), and cadmium (Cd) ions. It was found that Na-bonded γ-PGA with a high molecular weight showed the highest heavy metal adsorption (92.2-98.3%), particularly at a 0.5% concentration which exhibited the highest adsorption efficiency. Additionally, the study investigated the interaction of γ-PGA in mixed heavy metal environments, and it was discovered that Na-γ-PGA-HM at a 0.5% concentration showed a superior adsorption efficiency for Pb ions (85.4%), highlighting its selectivity as a potential effective biosorbent for wastewater treatment. This research not only enlightens the understanding of γ-PGA's role in heavy metal remediation but also underscores its potential as a biodegradable and non-toxic alternative for environmental cleanup. The findings pave the way for further exploration into the mechanisms and kinetics of γ-PGA's adsorption properties.

Cucumber (Cucumis sativus L.) with heterologous poly-γ-glutamic acid has skin moisturizing, whitening and anti-wrinkle effects.

Three genes involved in poly-γ-glutamic acid(γ-PGA)synthesis cloned from Bacillus licheniformis were transformed into cucumber for the first time. Compared with control, its water content increased by 6-14 % and water loss rate decreased by 11-12 %. In zebrafish and human skin experiments, the moisturizing effect of transgenic cucumber was significantly higher than that of CK, γ-PGA and hyaluronic acid group. Transgenic cucumber reduced facial wrinkles and roughness by 19.58 % and 24.97 %, reduced skin melanin content by 5.27 %, increased skin topological angle and L-value by 5.89 % and 2.49 %, and increased the R2 and Q1 values of facial elasticity by 7.67 % and 5.64 %, respectively. The expressions of aqp3, Tyr, silv and OCA2 were down-regulated, eln1, eln2, col1a1a and col1a1b were up-regulated in zebrafish after treated with transgenic cucumber. This study provides an important reference for the endogenous synthesis of important skin care functional molecules in plants.

Incorporation of poly(γ-glutamic acid) in lipid nanoparticles for enhanced mRNA delivery efficiency in vitro and in vivo.

Messenger RNA (mRNA)-based therapy shows immense potential for broad biomedical applications. However, the development of safe and efficacious mRNA delivery vectors remains challenging due to delivery barriers and inefficient intracellular payload release. Herein, we presented a simple strategy to boost the mRNA intracellular release by incorporation of anionic poly(γ-glutamic acid) (PGA) into an ionizable lipid-based LNP/mRNA. We systematically investigated the impact of PGA incorporation on mRNA transfection both in vitro and in vivo. The molecular weights and formulation ratios of PGA greatly affected the transfection efficacy of LNP/mRNA. From in vitro study, the optimized LNP/mRNA/PGA was formulated by incorporation of PGA with the molecular weight of 80 kDa or 200 kDa and the charge ratio (N/P/C) of 25/1/1. The optimized formulation achieved around 3-fold mRNA expression in HeLa cells compared to the bare LNP/mRNA. The intracellular releasing study using specific DNA probe revealed that this enhancement of transfection efficacy was attributed to the elevated mRNA release into cytoplasm. Moreover, the optimized LNP/mRNA/PGA achieved up to 5-fold or 3-fold increase of luciferase mRNA expression in vivo after being injected into mice systematically or intramuscularly, respectively. In addition, the incorporation of PGA did not significantly alter the biodistribution profile of the complexes on both organ and cellular levels. Therefore, our work provides a simple strategy to boost mRNA delivery, which holds great promise to improve the efficacy of mRNA therapeutics for various biomedical applications. STATEMENT OF SIGNIFICANCE: The process of designing and screening potent mRNA carriers is complicated and time-consuming, while the efficacy is not always satisfying due to the delivery barriers and inefficient mRNA release. This work presented an alternative strategy to boost the mRNA delivery efficacy by incorporating an anionic natural polymer poly(γ-glutamic acid) (PGA) into LNP/mRNA complexes. The optimized LNP/mRNA/PGA achieved up to 3-fold and 5-fold increase in transfection efficacy in vitro and in vivo, respectively. Intracellular releasing analysis revealed that the enhancement of transfection efficacy was mainly attributed to the elevated intracellular release of mRNA. In addition, the incorporation of PGA did not alter the biodistribution or the biosafety profile of the complexes. These findings indicate that PGA incorporation is a promising strategy to improve the efficacy of mRNA therapeutics.

Culture-dependent and -independent analyses of bacterial compositions and its contributions to formation of γ-aminobutyric acid and poly-γ-glutamic acid in Cheonggukjang.

This study was performed to unveil bacterial compositions and their contributions to the formation of γ-aminobutyric acid (GABA) and poly-γ-glutamic acid (γ-PGA) in Cheonggukjang. To predict possible key factors contributing to the content of the bioactive compounds in Cheonggukjang, commercial products were analyzed for various parameters. The content of GABA and γ-PGA showed a negative (R2 = 0.61 - 0.73) and positive correlation (R2 = 0.53 - 0.96) with antioxidative activity. Consistently, GABA content showed a moderate negative correlation with γ-PGA content (R2 = 0.58). Among the physicochemical and microbial parameters, only salinity showed a moderate negative correlation with γ-PGA content (R2 = 0.75), which might be due to the inhibition of bacterial growth. It was also suggested that multiple factors (including bacterial species) were involved in the formation of GABA and γ-PGA in Cheonggukjang. To reveal dominant bacterial species and further presume their contributions to the bioactive compound formation in Cheonggukjang, both culture-independent (metagenomic) and -dependent (culturomic) methods were used. Culture-independent method showed that Bacillus piscis was dominant (23.37 - 94.89 %), followed by B. hisashii (0.00 - 62.45 %) and B. coagulans (0.00 - 13.82 %). Considering the quantitative speciation data on the bioactive compound content in Cheonggukjang (and bacterial production capability) together, it was further elucidated that B. piscis contributed primarily to the bioactive compound formation. Unlike this, culture-dependent analysis revealed that B. licheniformis and B. subtilis were dominant (30.0 - 47.6 and 17.5 - 39.5 %, respectively). Based on the quantitative speciation data on the bacterial production capability of GABA and γ-PGA, B. subtilis was the primarily contributing bacterial species to the bioactive compound formation. Consequently, it was observed that the bacterial compositions and their contributions to the bioactive compound formation determined by the two methods differed considerably, i.e., B. piscis and B. subtilis were identified to be prominent bacterial contributors, respectively, depending on the method used.

Effects of γ-polyglutamic acid on grassland sandy soil properties and plant functional traits exposed to drought stress.

The current study provides field experimental data that support the use of γ-polyglutamic acid (γ-PGA) in drought stress and proposes its application in grassland management. We hypothesized that water treatment combined with PGA application to sandy soil would reduce drought stress in grasslands more effectively than watering alone. A randomized block design was used, with three replicate watering blocks (no watering, weekly watering, and monthly watering) and PGA treatments at four different concentrations (0%, 0.3%, 1%, and 2% PGA). The results showed that PGA acts as a biostimulant, alleviating the effects of stress in plants by: (1) increasing the availability of ions, especially K+, Zn2+, Mn2+, Fe2+/3+, Ca2+, and Mg2+, as well as N-NH4+, and N-NO3-, (2) elongating plant roots, (3) increasing the aboveground biomass, (4) improving the resprouting capacity of the dominant grass Nardus stricta, and (5) improving the regeneration of dicotyledons. In the case of meadows on sandy soils, the use of low PGA concentrations (0.3% or 1%) was the most beneficial for the availability of macro- and microelements and improving the functional traits of plants. Irrigation had a greater effect than using PGA only for the dicotyledon to monocotyledon ratio.

The influence of γ-PGA on the quality of cooked frozen crayfish during temperature fluctuations.

The purpose of this study was to compare the influences of gamma-poly glutamic acid (γ-PGA) (1, 2, 3, and 4 %) to see which could outperform conventional cryoprotectant mixture (4 % sorbitol + 4 % sucrose) on cooked crayfish properties, such as physicochemical, textural qualities, oxidation reaction, water distributions, and microstructure integrity, during different freeze-thaw cycles. Crayfish quality characteristics improved significantly as γ-PGA concentration increased compared to control samples.Adding γ-PGA 4 % reduced the carbonyl content from 4.20 to 3.00 nmol/ mg protein during fluctuation-1 (F1), and from 4.15 to 2.80 nmol/ mg protein during fluctuation-2 (F2) compared to control samples. Furthermore, it increased the total sulfhydryl content from 4.15 and 4.76 to 6.19 and 6.47 mol/105 g protein during F1 and F2 and after five freeze-thaw cycles (FTC). This suggests that this concentration was more effective at controlling protein changes than other concentrations. γ-PGA generally enhanced the water-holding capacity by preventing protein denaturation and limiting ice crystal recrystallization. As a result, microstructure stability was evident, texture degradation was avoided, and the crayfish's color was preserved.

Antidiabetic effects and mechanism of γ-polyglutamic acid on type II diabetes mice.

Diabetes is one of the foremost chronic non-communicable diseases worldwide, which significantly impacts people's quality of life. This study aimed to investigate the hypoglycemic effects of γ-polyglutamic acid (γ-PGA) on STZ-induced type II diabetes mice and its potential mechanisms. The results indicated that γ-PGA intervention contributed to reducing fasting blood glucose levels in diabetic mice, regulating lipid metabolism in type II diabetes mice, and improving insulin resistance. Additionally, γ-PGA could alleviate liver inflammation, enhancing the activity of hepatic antioxidant enzymes. Investigation into the insulin signaling pathway revealed that γ-PGA significantly increased the expression of INSR, IRS-1, Akt, PI3K in diabetic mice, thereby enhancing insulin sensitivity and improving insulin resistance to regulate glucose metabolism. High-throughput sequencing of mouse gut microbiota using 16S rRNA showed that γ-PGA increased the abundance and evenness of beneficial bacteria in the intestines of type II diabetic mice, inhibited the growth of harmful bacteria, and may exerted hypoglycemic effects by modulating and improving relevant metabolic pathways associated with diabetes symptoms. This study provides new insights into the treatment of type II diabetes and highlights the significant potential of γ-PGA in treating type II diabetes.

An injectable bioactive poly(γ-glutamic acid) modified magnesium phosphate bone cement for bone regeneration.

As potential alternatives for calcium phosphate bone cements, magnesium phosphate bone cements (MPC) have attracted considerable attention in recent years. However, their several defects, such as rapid setting times, highly hydration temperature and alkaline pH due to the part of the unreacted phosphate, restricted their applications in human body. With aim to overcome these defects, a novel polypeptite poly(γ-glutamic acid) (γ-PGA) modified MPC were developed. Effect of γ-PGA content on the injectability, anti-washout ability, setting times, hydration temperature, mechanical compressive strength, in vitro bioactivity and degradation were investigated. Moreover, in vitro cyto-compatibility was evaluated using MC3T3-E1 cells by CCK-8 and Live/Dead staining. All these results indicated that the 10%PGA-MPC with an improved handling performances, low hydration temperature, high mechanical compressive strength, and good cyto-compatibility hold a great potential for bone repair and regeneration.

Glutamic Acid and Poly-γ-glutamic Acid Enhanced the Heat Resistance of Chinese Cabbage (Brassica rapa L. ssp. pekinensis) by Improving Carotenoid Biosynthesis, Photosynthesis, and ROS Signaling.

Heat stress is one of the most common agrometeorological risks in crop production in the middle and lower reaches of the Yangtze River in China. This study aimed to investigate whether glutamic acid (Glu) or poly-γ-glutamic acid (γ-PGA) biostimulants can improve the thermotolerance of a cool-season Chinese cabbage (Brassica rapa L. ssp. pekinensis) crop. Priming with Glu (2.0 mM) or γ-PGA (20 mg·L-1) was conducted at the third leaf stage by applying as daily foliar sprays for 5 days before 5 days of heat stress (45 °C in 16-h light/35 °C in 8-h dark). Coupled with morpho-physiological and biochemical analyses, transcriptomes of Glu or γ-PGA-primed Chinese cabbage under heat stress were examined by RNA-seq analysis. The results showed that the thermotolerance conferred by Glu and γ-PGA priming was associated with the increased parameters of vegetative growth, gas exchange, and chlorophyll fluorescence. Compared with the control, the dry weights of plants treated with Glu and γ-PGA increased by 51.52% and 39.39%, respectively. Glu and γ-PGA application also significantly increased the contents of total chlorophyll by 42.21% and 23.12%, and carotenoid by 32.00% and 24.00%, respectively. In addition, Glu- and γ-PGA-primed plants markedly inhibited the levels of malondialdehyde, electrolyte leakage, and super-oxide anion radical, which was accompanied by enhanced activity levels of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and peroxidase (POD). Enrichment analysis of Kyoto Encyclopedia of Genes and Genomes (KEGG) categories within the differentially expressed genes (DEGs) functional clusters of RNA-seq data indicated that the expression levels of the genes for DNA replication, DNA repair system, linoleic acid metabolism, cysteine and methionine metabolism, glutathione metabolism, purine and pyrimidine metabolism, carotenoid biosynthesis, and plant-pathogen interaction were commonly up-regulated by both Glu and γ-PGA priming. Glu treatment enhanced the expression levels of the genes involved in aliphatic glucosinolate and 2-oxocarboxylic acid, while γ-PGA treatment activated carotenoid cleavage reaction to synthesize abscisic acid. Taken together, both Glu and γ-PGA have great potential for the preadaptation of Chinese cabbage seedlings to heat stress, with Glu being more effective than γ-PGA.

The role of structural evolution in the complexation and flocculation of heavy metals by the microbial product poly-γ-glutamic acid.

The process and mechanism of heavy metal flocculation with extracellular polymeric substances (EPS) secreted by microorganisms, are crucial to their fate in natural environment, wastewater treatment and soil bioremediation applications. However, the structural features of EPS and the relationship between these features and the flocculation process and mechanism remain unclear. In the present study, structural features of the microbial product poly-γ-glutamic acid (γ-PGA) complexed with the heavy metal ions Pb2+ and Cu2+ were characterized and the evolution of these features was identified as having a key role in the flocculation process and mechanism. The secondary structure of the γ-PGA-Pb complex changed significantly, while that of the γ-PGA-Cu complex was only slightly altered. The significant structural change in γ-PGA-Pb was found to be responsible for the combination of residual COOH and Pb2+, promoting the bridging of inter-colloids and faster growth of hydrodynamic diameter. If the conformation changed sufficiently, such as with the γ-PGA-Pb complex in the pH range 4.6-6.2, pH had no impact on the conversion ratio. The unchanged structure of γ-PGA-Cu prevented the flocculation process, although the coordination mode of γ-PGA-Cu resulted in a higher biosorption capacity. This in-depth molecular-level study provides insight into the γ-PGA flocculation mechanism, promoting the use of γ-PGA and γ-PGA producing microorganisms for application in various remediation strategies.

Structural and genetic insights into a poly-γ-glutamic acid with in vitro antioxidant activity of Bacillus velezensis VCN56.

Poly-γ­glutamic acid (γ­PGA) produced by Bacillus species is a natural biopolymer, which is widely used in various fields including food, pharmaceuticals, and cosmetics. In this study, the screening of 19 Bacillus isolates derived from traditionally fermented foods revealed that Bacillus velezensis VCN56 was the most potent γ­PGA producer. The maximum concentration of crude γ­PGA was 32.9 ± 1.5 g/L in the PGA-3 medium containing glycerol, citric acid, sodium glutamate, NH4Cl, and starch. The resulting γ-PGA was purified and then characterized by HPLC, FTIR, and 1H-NMR analyses. Molecular weight of purified γ­PGA was estimated to be 98 kDa with a polydisperse index of 2.04. Notably, the pure γ­PGA showed significant in vitro antioxidant scavenging activities against 1,1-diphenyl-2-picrylhydrazyl (72.0 ± 1.5%), hydroxyl (81.0 ± 0.6%), and superoxide (43.9 ± 0.8%) radicals at the concentration of 4 mg/mL. Using whole-genome sequencing, the genetic organization of pgs operon responsible for γ­PGA biosynthesis in B. velezensis VCN56 differs from those in other Bacillus genomes. Further genome analysis revealed metabolic pathways for γ-PGA production and degradation. For the first time, the present study provides a better understanding of γ-PGA with a promising antioxidant activity produced by B. velezensis at the phenotypic, biochemical, and genomic levels, which hold potential applications in the foods, cosmetics, and pharmaceutical industries.

Genetic and metabolic engineering for poly-γ-glutamic acid production: current progress, challenges, and prospects.

Accompanied with the developments of gene editing and synthetic biology toolkits, various metabolic engineering strategies have been established for strain improvement to enhance the target metabolite production. Poly-γ-glutamic acid (γ-PGA) is a natural biopolymer that mainly produced by Bacillus, and low-level yield hinders its application. To address this problem, numerous approaches have been conducted to increase γ-PGA yield. In this review, we focus on the genetic and metabolic engineering of microorganism for γ-PGA production, including strengthening raw materials utilization and precursor supply, enhancing γ-PGA synthetase gene cluster, transcription regulation engineering, cofactor regeneration, energy engineering and blocking the synthetic pathways of by-products. Meanwhile, to attain the γ-PGA with different configurations (D/L) and molecular weights, the expression of γ-PGA synthetase, glutamate racemase and γ-PGA hydrolase were respectively manipulated. In addition, except for Bacillus, metabolic engineering of other hosts for high-level production of γ-PGA was also reviewed in this article. Finally, the prospect of metabolic engineering of γ-PGA production strain was discussed regarding the recent progress, challenge, and trends in this field.

Membrane stabilization versus perturbation by aromatic monoamine-modified γ-PGA for cryopreservation of human RBCs with high intracellular trehalose.

As a nonreducing disaccharide, trehalose can be used as a biocompatible cryoprotectant for solvent-free cell cryopreservation, but the membrane-impermeability limits its cryoprotective efficiency. Herein, a series of aromatic monoamines with a 1-4 methylene spacer were grafted onto γ-poly(glutamic acid) (γ-PGA) for promoting intracellular trehalose uptake in human red blood cells (hRBCs) via membrane perturbation. The self-assembled nanoparticles of the obtained amphiphilic γ-PGA could be adsorbed on the cell membrane by the hydrophobic interaction to disturb the lipid arrangement and increase the membrane permeability of trehalose under hypertonic conditions. Results suggested that the intracellular trehalose could be enhanced progressively with the methylene spacer length, significantly increasing to 75.1 ± 0.7 mM by incubating hRBCs in 0.8 M trehalose containing phenylbutylamine-grafted γ-PGA at 4 °C for 24 h. Meanwhile, the other three polymers exhibited membrane stabilization in addition to improved intracellular trehalose, maintaining the membrane integrity during cryopreservation to achieve high cryosurvival. Molecular dynamics simulation further confirmed that defects could be formed by interaction of the above four amphiphilic polymers on the modeled phospholipid bilayer. It was believed that glycerol-free cryopreservation of human cells could be realized by using trehalose as the biocompatible cryoprotectant, and membrane stabilization can be a compensatory approach to membrane perturbation during impermeable biomolecule delivery.

Efficient molasses utilization for low-molecular-weight poly-γ-glutamic acid production using a novel Bacillus subtilis stain.

BACKGROUND: Poly-γ-glutamic acid (γ-PGA) is a biopolymer and has various applications based on its biocompatibility, non-toxicity, and edibility. Low-molecular-weight (Mw)-γ-PGA has promising applications in agriculture and pharmaceuticals. It is traditionally produced by enzymatic hydrolysis. Cost-effective bioproduction of low-Mw-γ-PGA is essential for commercial application of γ-PGA. RESULTS: Bacillus subtilis 242 is a newly isolated low-Mw-γ-PGA-producing strain. To develop cost-effective production of γ-PGA using this newly isolated strain, cane molasses and corn steep liquor were used to produce γ-PGA. The concentration of cane molasses was optimized and 100 g/L cane molasses resulted in high γ-PGA production. The effects of yeast extract and corn steep liquor on γ-PGA yield were investigated. High concentration of γ-PGA was obtained in the medium with corn steep liquor. A concentration of 32.14 g/L γ-PGA was achieved in fed-batch fermentation, with a productivity of 0.67 g/L/h and a percentage yield (gγ-PGA/gglutamate) of 106.39%. The Mw of γ-PGA was 27.99 kDa. CONCLUSION: This study demonstrated the potential application of B. subtilis 242 for cost-effective production of low-Mw-γ-PGA from cane molasses.

Quantitation of methotrexate polyglutamates in human whole blood, erythrocytes and leukocytes collected via venepuncture and volumetric absorptive micro-sampling: a green LC-MS/MS-based method.

Low-dose methotrexate (MTX) plays a key role in treatment of rheumatoid arthritis. However, not all patients respond satisfactorily, and no therapeutic drug monitoring has been implemented in clinical practice, despite the fact that MTX therapy has now been available for decades. Analysis of individual intracellular MTX metabolites among rheumatoid arthritis (RA) patients is hampered by the low intracellular concentrations of MTX-PGs which require a highly sensitive method to quantify. Here, we present a rapid and highly sensitive LC (HILIC) MS/MS method with LLOQ 0.1 nM, 0.8 nmol/L for each metabolite of MTX-PG1-5 and MTX-PG6-7 respectively. Over a linear range of 0.1-100 nM, 0.8-100 nmol/L for each metabolite of MTX-PG1-5 and MTX-PG6-7, respectively, the inter- and intra- accuracy and precision were within 15% of the nominal value for all MTX metabolites. The presented assay was used to assess and compare MTX metabolite concentrations extracted from four different matrices: red blood cells, plasma, peripheral blood mononuclear cells, and whole blood that have been collected either using traditional venepuncture or volumetric absorptive micro-sampling (VAMS) sampling techniques. The presented method not only improves analyte coverage and sensitivity as compared to other published methods; it also improves the greenness.

γ-Polyglutamic Acid Production, Biocontrol, and Stress Tolerance: Multifunction of Bacillus subtilis A-5 and the Complete Genome Analysis.

Bacillus subtilis A-5 has the capabilities of high-molecular-weight γ-PGA production, antagonism to plant pathogenic fungi, and salt/alkaline tolerance. This multifunctional bacterium has great potential for enhancing soil fertility and plant security in agricultural ecosystem. The genome size of B. subtilis A-5 was 4,190,775 bp, containing 1 Chr and 2 plasmids (pA and pB) with 43.37% guanine-cytosine content and 4605 coding sequences. The γ-PGA synthase gene cluster was predicted to consist of pgsBCA and factor (pgsE). The γ-PGA-degrading enzymes were mainly pgdS, GGT, and cwlO. Nine gene clusters producing secondary metabolite substances, namely, four unknown function gene clusters and five antibiotic synthesis gene clusters (surfactin, fengycin, bacillibactin, subtilosin_A, and bacilysin), were predicted in the genome of B. subtilis A-5 using antiSMASH. In addition, B. subtilis A-5 contained genes related to carbohydrate and protein decomposition, proline synthesis, pyruvate kinase, and stress-resistant proteins. This affords significant insights into the survival and application of B. subtilis A-5 in adverse agricultural environmental conditions.

Construction of a mutant Bacillus subtilis strain for high purity poly-γ-glutamic acid production.

OBJECTIVE: To construct a Bacillus subtilis strain for improved purity of poly-γ-glutamic acid. RESULTS: The construction of strain GH16 was achieved by knocking out five genes encoding extracellular proteins and an operon from Bacillus subtilis G423. We then analyzed the amount of protein impurities in the γ-PGA produced by the resulting strain GH16/pHPG, which decreased from 1.48 to 1.39%. Subsequently the fla-che operon, PBSX, as well as the yrpD, ywoF and yclQ genes were knocked out successively, resulting in the mutant strains GH17, GH18 and GH19. Ultimately, the amount of protein impurities was reduced from 1.48 to 0.83%. In addition, the amount of polysaccharide impurities in the γ-PGA was also decreased from 2.21 to 1.93% after knocking out the epsA-O operon. CONCLUSIONS: The high purity γ-PGA producer was constructed, and the resulting strain was a promising platform for the manufacture of other highly pure extracellular products and secretory proteins.